DIETARY RESTRICTION OF ISOLEUCINE INCREASES THE HEALTHSPAN AND LIFESPAN OF GENETICALLY HETEROGENEOUS MICE

Abstract Low protein (LP) diets promote health and longevity in diverse species, andreducing dietary levels of the branched-chain amino acids (BCAAs) leucine, isoleucine and valine recapitulates these effects in C57BL/6J mice, promoting metabolic health in both sexes, and increases lifespan while reducing frailty in males. Each BCAA has unique metabolic effects, and we recently showed that restriction of isoleucine is both sufficient to promote metabolic health and required for the metabolic benefits of an LP diet in C57BL/6J males. Here, we tested the hypothesis that specifically restricting dietary isoleucine could promote healthy aging in adult genetically heterogenous UM-HET3 mice. We find that isoleucine restriction (IleR) improves the metabolic health of both young and old HET3 mice, promoting leanness and glycemic control in both sexes, and reprograms hepatic metabolism, blunting age-related molecular changes in males and, to a lesser extent, in females. Finally, we find that IleR reduces frailty and extends both the median and maximum lifespan of both male and female HET3 mice, but to a much greater degree in males. Our results demonstrate that restricting dietary isoleucine can increase health span and longevity in a genetically diverse population of mice and suggest that reducing dietary levels of isoleucine, or drug regimens that mimic this effect, may be a novel intervention to promote healthy aging.

be effective in delaying AD progression, however, inexpensive and non-invasive markers for routine laboratory examination are poorly developed.Studies indicate that poor oral health is associated with a higher risk of dementia via microbiome alteration.In this pilot study, we explored the importance of the multiomics analysis in discriminating cognitively healthy from mild cognitive impaired and dementia by comparing the microbiome and proteomic profiles of saliva from controls (n=37), MCI (n=16) and dementia (n=6) participants of the MiaGB (Microbiome in aging Gut and Brain) consortium.We detected significantly reduced abundance of oral commensals like B. animalis, E. brachy, and F. fastidiosum in MCI and dementia samples than controls with specific increase of P. histicola in MCI and neuronal Aβ1-42 promoting V. parvula in the dementia.In addition, proteome analyses revealed that highly neuronal relevant nitrite-nitrate conversion cycle genes were significantly differed in dementia and MCI than controls.Discriminatory predictive analyses revealed that combination of microbiome and proteome signature are stronger discriminator for dementia/MCI from cognitively healthy controls.Our results indicate the potential links among oral microbiome, proteome and dementia, as well as diagnostic potential for dementia risk.

DIETARY RESTRICTION OF ISOLEUCINE INCREASES THE HEALTHSPAN AND LIFESPAN OF GENETICALLY HETEROGENEOUS MICE Cara Green, University of Wisconsin-Madison, Madison, Wisconsin, United States
Low protein (LP) diets promote health and longevity in diverse species, andreducing dietary levels of the branchedchain amino acids (BCAAs) leucine, isoleucine and valine recapitulates these effects in C57BL/6J mice, promoting metabolic health in both sexes, and increases lifespan while reducing frailty in males.Each BCAA has unique metabolic effects, and we recently showed that restriction of isoleucine is both sufficient to promote metabolic health and required for the metabolic benefits of an LP diet in C57BL/6J males.Here, we tested the hypothesis that specifically restricting dietary isoleucine could promote healthy aging in adult genetically heterogenous UM-HET3 mice.We find that isoleucine restriction (IleR) improves the metabolic health of both young and old HET3 mice, promoting leanness and glycemic control in both sexes, and reprograms hepatic metabolism, blunting age-related molecular changes in males and, to a lesser extent, in females.Finally, we find that IleR reduces frailty and extends both the median and maximum lifespan of both male and female HET3 mice, but to a much greater degree in males.Our results demonstrate that restricting dietary isoleucine can increase health span and longevity in a genetically diverse population of mice and suggest that reducing dietary levels of isoleucine, or drug regimens that mimic this effect, may be a novel intervention to promote healthy aging.

EAAS AND WHEY PROTEIN MAY IMPROVE PHYSICAL FUNCTION OF OLDER ADULTS INDEPENDENTLY OF GUT MICROBIOME
Gohar Azhar, Ambika Verma, Intawat Nookaew, Pankaj Patyal, Shakshi Sharma, Robert Wolfe, and Jeanne Wei, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States Essential amino acids (EAAs) and dietary proteins are major nutritional supplements that support the growth and activity of gut microbes, contributing to the wellbeing of their host.We hypothesized that daily ingestion of EAAs or whey proteins for twelve weeks would improve the gut microbiome of older adults and thereby have a positive impact on their physical functioning.The stool samples were collected from the 40 subjects before and after consuming EAAs or whey proteins for twelve weeks and subjected to Illumina based 16S rRNA gene sequencing.In both groups, the most abundant families in order of relative abundance included: Bacteroidaceae, Ruminococcaceae, Lachnospiraceae, Rikenellaceae, and Prevotellaceae, which indicated that these subjects were able to maintain a dominant consortium of microbes in their guts that have been associated with healthy aging.Individual gut microbiome samples analysis with nonmetric multidimensional space (NMDS) showed that each individual was associated with its unique microbiome.
The key finding of this study was a significant reduction of plasma Interleukin-18 binding protein (IL-18) in EAAs vs whey group (p < 0.003).It is plausible that the reduced level of inflammatory IL-18 contributed to improved intestinal immunity.Moreover, the reduced inflammation might have resulted in better muscle performance in the EAAs group vs whey as evidenced by six-minute walking distance (EAAs 92.55 ± 4.16 ft vs whey 47.46 ± 4.99 ft, p< 0.0001) at the completion of study.Future studies will need to explore the role of EAAs on gut microbiome mediated reduction of immune inflammation on physical function and aging.

IDENTIFYING SENOLYTIC TARGETS OF DIETARY FLAVONOIDS IN RENAL CELLS BY THERMAL PROTEOME PROFILING
Quinn Strassheim, Reema Banarjee, Delaney Rutherford, Amit Dey, Dimitrios Tsitsipatis, Ruin Moaddel, Myriam Gorospe, and Nathan Basisty, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States Senescent cell accumulation is a known driver of aging and age-related pathologies.Clearance of senescent cells is a promising approach to increase longevity and reduce multiple age-related diseases in humans.Flavonoid compounds are present in fruits and vegetables, of which certain flavonoids (e.g., quercetin and fisetin) have demonstrated senolytic, or 'senescent-cell-killing', activity in culture and in mice.However, the cellular mechanism of senolysis and the efficacy of these compounds are not known in renal epithelial cells.Here, we combine flavonoid drug screening, targeted metabolomics, and thermal proteome profiling (TPP) with analysis by mass spectrometry (MS) to explore protein targets of senolytic drugs in senescent human renal cortical epithelial cells and human renal proximal tubular epithelial cells.
Senescence was induced by exposure to ionizing radiation and the senescence phenotype was validated through a rigorous panel of senescence and viability markers.Proliferating and senescent renal epithelial cells were screened with a panel of 8 flavonoids to identify senolytic drugs and their concentrations for selectively killing senescent cells.Targeted MS assays were developed to assess intracellular drug uptake, a potential mechanism of senescent-specific killing.To identify protein targets of the senolytic flavonoids in senescent cells, we performed a variation of TPP in combination with liquid chromatography (LC)-MS/MS proteomics analysis, carefully controlling for non-senolytic interactions by excluding proteins bound by a non-senolytic flavonoid.The results of this study pave the way for the development of a more specific generation of senolytic compounds and identify novel candidate senolytic pathways engaged by dietary flavonoids.

LIVING LONGER WITH MODERATE AMOUNTS OF OXYGEN RADICALS IN FRUIT FLIES
Man Su Kim, and Saurav Ghimire, Inje University, Gimhae,

Republic of Korea
The role of reactive oxygen species (ROS) in aging is complex and paradoxical.While the free radical theory of aging suggests that ROS cause cellular damage and senescence, some studies have shown that moderate ROS levels can enhance cellular adaptation and longevity.In this research, we used Drosophila as an in vivo model to investigate the optimal level of ROS that can promote healthy aging.We exposed flies to different concentrations of paraquat (PQ), a herbicide that generates superoxide anions, and measured their lifespan, stress resistance, and gene expression.We found that intermediate PQ levels (0.1 mM) extended lifespan and improved stress tolerance, while high PQ levels (0.5 mM) shortened lifespan and impaired stress response.We also observed an up-regulation of ROS scavenger genes, such as superoxide dismutase 1 and glutathione peroxidase, in the intermediate PQ group, suggesting a homeostatic mechanism to balance ROS levels.Our findings challenge the conventional view of ROS as harmful agents and provide novel insights into the molecular mechanisms of aging.Branched-chain amino acids (BCAAs) are pivotal for the health of the human body.Leucine, isoleucine, and valine, which make up the BCAAs, are α-amino acids that promote skeletal muscle growth and development.Valine is one of the essential BCAAs required for the synthesis of muscle proteins and the support growth of skeletal muscles.Valine also plays a favorable role in enhancing insulin sensitivity, preserving intestinal health, and optimizing lipid metabolism.Considering the relevance of valine in promoting muscle growth and metabolism, we utilized C2C12 skeletal muscle cell line to investigate the role of valine in regulating mitochondrial functions.C2C12 cells grown on physiological normal glucose media (100mg/dL) were used